The invention is in the field of recombinant genetics.
Both viral and cloned reverse transcriptase (RT) contain at least two enzymatic activities, DNA polymerase and ribonuclease H (RNase H), that reside on a single polypeptide. Grandgenett, D. P. et al., Proc. Natl. Acad. Sci. (USA) 70:230-234 (1973); Moelling, K., Virology 62:46-59 (1974); Kotewicz, M. L., et al., Gene 35:249-258 (1985); and Roth, M. J., et al., J. Biol. Chem. 260:9326-9335 (1985). Little is known about the structure-functional relationship of these two activities, but such knowledge would be important both in understanding retroviral replication and in exploiting the enzyme as a recombinant DNA tool.
In the retrovirus life cycle, the RT DNA polymerase activity is responsible for transcribing viral RNA into double-stranded DNA. Varmus, H. (1982), in Weiss, R., et al. (eds.), RNA Tumor Viruses, Cold Spring Harbor Laboratory, pp. 410-423. The function of RNase H in replication is less clear, but it is thought to degrade genomic RNA during DNA synthesis to generate oligomeric RNA primers for plus-strand DNA synthesis, and to remove the RNA primers of both minus- and plus-strand DNA. Omer, C. A., et al., Cell 30:797-805 (1982); Resnick, R., et al., J. Virol. 51:813-821 (1984); Varmus, H. (1985), in Weiss, R., et al. (eds.), RNA Tumor Viruses, Cold Spring Harbor Laboratory, pp. 79-80.
The temporal relationship in vivo between DNA polymerization and RNA hydrolysis is not well defined. Furthermore, precisely how the two enzymatic activities are coordinated is not clear. Conditional mutations restricted to either DNA polymerase or RNase H would be invaluable in deciphering the events of retroviral replication. Unfortunately, conditional viral mutations in the RT gene invariably affect both activities. Lai, M. H. T, et al., J. Virol. 27:823-825 (1978); Moelling, K., et al., J. Virol. 32:370-378 (1979).
RT is used extensively in recombinant DNA technology to synthesize cDNA from mRNA. One major problem with cDNA synthesis is that the RNase H activity of RT degrades the mRNA template during first-strand synthesis. The mRNA poly(A)-oligo(dT) hybrid used as a primer for first-strand cDNA synthesis is degraded by RT RNase H. Thus, at the outset of cDNA synthesis, a competition is established between RNase H-mediated deadenylation of mRNA and initiation of DNA synthesis, which reduces the yield of cDNA product. Berger, S. L., et al., Biochem. 22:2365-2373 (1983). Furthermore, in some cases, the RNase H causes premature termination of DNA chain growth. Unfortunately, these events eliminate the potential for repeated copying of the RNA template.
Efforts to selectively inactivate RT RNase H with site-specific inhibitors have been unsuccessful (for review, see Gerard, G. F. (1983), in Jacob, S. T., (ed.), Enzymes of Nucleic Acid Synthesis and Modification, Vol. I, DNA Enzymes, CRC Press, Inc., Boca Raton, Fla., pp. 1-38). Attempts to physically separate the active centers of RT polymerase and RNase H activity by proteolysis have yielded a proteolytic fragment possessing only RNase H activity (Lai, M. H. T., et al., J. Virol. 25:652-663 (1978); Gerard, G. F., J. Virol. 26:16-28 (1978); and Gerard, G. F., J. Virol. 37:748-754 (1981)), but no corresponding fragment containing only polymerase activity has been isolated.
Computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment at the carboxyl terminus that is homologous with the ribonuclease H of E. coli and a section close to the amino terminus which can be aligned with nonretroviral polymerases. Johnson, M. S., et al., Proc. Natl. Acad. Sci. (USA) 83:7648-7652 (1986). Based on these related amino acid sequences, Johnson et al. suggest that ribonuclease H activity should be situated at the carboxyl terminus, and the DNA polymerase activity at the amino terminus.
There have been a number of reports concerning the cloning of genes which encode RT and their expression in hosts. Weiss et al., U.S. Pat. No. 4,663,290 (1987); Gerard, G. F., DNA 5:271-279 (1986); Kotewicz, M. L., et al., Gene 35:249-258 (1985); Tanese, N., et al., Proc. Natl. Acad. Sci. (USA) 82:4944-4948 (1985); and Roth, M. J., et al., J. Biol. Chem. 260:9326-9335 (1985).
The invention relates to a gene which encodes reverse transcriptase having DNA polymerase activity and substantially no RNase H activity.
The invention also relates to a reverse transcriptase gene comprising the following DNA sequence:
or the degenerate variants thereof.
The invention also relates to a reverse transcriptase gene comprising the following DNA sequence:
or the degenerate variants thereof.
The invention also relates to the vectors containing the gene of the invention, hosts transformed with the vectors of the invention, and the reverse transcriptase expressed by the transformed hosts of the invention.
The invention also relates to a fusion protein comprising a polypeptide having RNA-dependent DNA polymerase activity and substantially no RNase H activity and a second peptide selected from polypeptide proteins which stabilize the fusion protein and hydrophobic leader sequences.
The invention also relates to a method of producing reverse transcriptase having DNA polymerase activity and substantially no RNase H activity, comprising culturing transformed hosts of the invention under conditions which produce reverse transcriptase, and isolating the reverse transcriptase so produced.
The invention also relates to a method of preparing cDNA from mRNA comprising contacting mRNA with a polypeptide having RNA-dependent DNA polymerase activity and substantially no RNase H activity, and isolating the cDNA so produced.
The invention also relates to a kit for the preparation of cDNA from mRNA comprising a carrier being compartmentalized to receive in close confinement therein one or more containers, wherein
(a) a first container contains reverse transcriptase having DNA polymerase activity and substantially no RNase H activity;
(b) a second container contains a buffer and the nucleoside triphosphates;
(c) a third container contains oligo(dT)primer; and
(d) a fourth container contains control RNA.
The invention is related to the discovery that portions of the RT gene can be deleted to give a deletion mutant having DNA polymerase activity but no detectable RNase H activity. This purified mutant RT lacking RNase H activity can be used to effectively synthesize cDNA from mRNA.